Disulfide Bond Formation in the Cytoplasm

Abstract

Significance: Disulfide bond formation is critical for biogenesis of many proteins. While most studies in this field are aimed at elucidating the mechanisms in the endoplasmic reticulum, intermembrane space of mitochondria, and prokaryotic periplasm, structural disulfide bond formation also occurs in other compartments including the cytoplasm. Such disulfide bond formation is essential for biogenesis of some viruses, correct epidermis biosynthesis, thermal adaptation of some extremophiles, and efficient recombinant protein production. Recent Advances: The majority of work in this new field has been reported in the past decade. Within the past few years very significant new data have emerged on the catalytic and noncatalytic mechanisms for disulfide bond formation in the cytoplasm. This includes the crystal structure of a key component of viral oxidative protein folding, identification of a missing component in cytoplasmic disulfide bond formation in hyperthermophiles, and introduction of de novo dithiol oxidants in engineered oxidative folding pathways. Critical Issues and Future Directions: While a broad picture of cytoplasmic disulfide bond formation has emerged many critical questions remain unanswered. The individual components in the natural systems are largely known, but the molecular mechanisms by which these processes occur are largely deduced from the mechanisms of analogous components in other compartments. This prevents full understanding and manipulation of these systems, including the potential for novel anti-viral drugs based on the unique features of their sulfhydryl oxidases and the generation of more efficient cell factories for the large-scale production of therapeutic and industrial proteins. Antioxid. Redox Signal. 19, 46–53.

Introduction

F or many years the formation of disulfide bonds, covalent linkages between the side chains of cysteine residues in proteins, was thought to occur only in proteins destined for secretion or the outer membrane. Such proteins fold in the periplasm of prokaryotes or the endoplasmic reticulum (ER) of eukaryotes and hence, in these compartments there exist extensive machinery for the formation of native disulfide bonds [reviewed and compared in (10)]. Around a decade ago, catalytic machinery for disulfide formation was found in the intermembrane space (IMS) of mitochondria [reviewed in (49)] and there have been sporadic reports connected with disulfide bond formation in chloroplasts [reviewed elsewhere in this issue (26)]. Given the evolution of eukaryotic organisms it is perhaps unsurprising that such machinery exists. For example, IMS of mitochondria is evolutionarily related to the periplasm of the free-living prokaryote from which the symbiosis arose.

The one major cellular compartment that is not often discussed with regard to disulfide bond formation is the cytoplasm. However, multiple natural and engineered pathways exist for disulfide bond formation in this compartment. This review will focus on what is known and what is unknown about the cellular mechanisms for the natural pathways (including those arising from metabolic processes, catalyzed viral protein biogenesis, keratinization, and catalyzed protein folding in some hyperthermophiles), and on efforts to engineer oxidative folding pathways into the cytoplasm, primarily into the cytoplasm of Escherichia coli.

The cytoplasm is a reducing environment

Before considering the pathways for disulfide bond formation in the cytoplasm we should examine the normal redox state of this compartment. The cytoplasm is usually stated to be a reducing environment, an environment in which disulfide bonds are broken rather than formed. However, disulfide bonds may be formed here, for example, in the catalytic cycle of class I and class II ribonucleotide reductases, one disulfide bond is formed in the active site of the enzyme for each deoxynucleotide formed [reviewed in (28)]. Similarly, disulfide bonds may be formed between two molecules of reduced glutathione to form oxidized glutathione as part of its antioxidant action [reviewed in (39)] or within a protein due to the nonspecific action of reactive oxygen species (ROS), including molecular oxygen or the nonspecific action of other reactive chemical species such as dehydroascorbate (45). Once disulfide bonds are formed in the cytoplasm there exist two distinct pathways for reducing them (Fig. 1). Both pathways ultimately use NADPH and hence, maintaining the reducing environment is an energy-dependent process.

FIG. 1.

Interlinked pathways for disulfide bond reduction in the cytoplasm of Escherichia coli. Two pathways for disulfide bond reduction exist in the cytoplasm of E. coli. One is based on the action of the abundant tripeptide glutathione and glutathione reductase. The other is based on the action of one of the two members of the thioredoxin family combined with that of thioredoxin reductase. Both glutathione reductase and thioredoxin reductase use NADPH. While these two pathways are often drawn as being parallel, thioredoxin superfamily members can be reduced by reduced glutathione and hence there is functional crosstalk between the two. Arrows indicate electron transfer.

Some of the mechanisms by which structural disulfide bonds can form in the cytoplasm are based on compromising these reducing pathways (see the sections on Keratinization and Engineered oxidative folding pathways) while others are based on catalyzed processes that coexist with the reducing pathways, the best known example of which is viral protein biogenesis.

Viral protein biogenesis

Disulfide bond formation in the ER and mitochondrial IMS is thought to be primarily based on the action of sulfhydryl oxidases, enzymes that use molecular oxygen to catalyze de novo disulfide bond formation (Fig. 2). There are multiple types of sulfhydryl oxidase, but they can be broadly broken down in two classes, the Ero [ER oxidase; reviewed in (51)] family members and the Erv/ALR [“essential for respiration and viability”/”augmenter of liver regeneration”; reviewed in (12)] family members. Ero and Erv family members are found in the eukaryotic secretory pathway, for example, in humans Ero1α, Ero1β, and quiescin-sulfhydryl oxidase [reviewed in (27, 51)]; while disulfide bond formation in the IMS of mitochondria is linked to Erv family members, for example, Erv1p in Saccharomyces cerevisae [reviewed in (49)]. Most eukaryotic sulfhydryl oxidase family members are thought not to directly oxidize folding proteins, rather they oxidize an intermediate protein, protein disulfide isomerase [PDI; reviewed in (21)] in the ER or Mia40 [reviewed in (49)] in the IMS; though in some cases such as quiescin-sulfhydryl oxidase the two proteins are fused [reviewed in (27)].

FIG. 2.

Parallels between disulfide bond formation by viruses in the cytoplasm and endogenous pathways in other eukaryotic cellular compartments. In endogenous pathways for eukaryotic disulfide bond formation there is an intermediary protein between the sulfhydryl oxidase, which de novo generates disulfide bonds, and the folding protein. In the endoplasmic reticulum (ER) (left panel) the intermediate protein, a protein disulfide isomerase (PDI)-family member, is oxidized by an Ero family member [reviewed in (51) along with regulation of this process]. Similarly, in the intermembrane space (IMS) of mitochondria (center panel) the Erv-family member oxidizes the intermediary protein Mia40 [reviewed in (49); note Erv1p may also transfer electrons to cytochrome c as well as to molecular oxygen; (5)]. This arrangement may allow greater regulation/control of these essential processes. This arrangement also holds in viruses (right panel), where the sulfhydryl oxidase for example, vaccinia virus E10R that exists in a complex with A2.5L (47) or African swine fever virus pB119L (16), appears to use an intermediary protein for example, G4L or pA151R respectively [reviewed in (17)]. In each case where molecular oxygen is the ultimate electron acceptor it is reduced to hydrogen peroxide. The peroxide produced by sulfhydryl oxidases may also be utilized via noncatalytic (24) or catalytic [e.g. (36, 55)] mechanisms to generate disulfide bonds. Arrows indicate electron transfer.

Many viruses use or adapt the folding systems in the host, for example, those found in the ER [reviewed in (6)]. However, more than a decade ago it was discovered that some viruses encoded their own oxidative folding pathway that catalyzed disulfide bond formation in the cytoplasm. The molecular pathway for this process was first elucidated for vaccinia virus (47) and the pathway shares features with the pathways found in the ER and IMS (Fig. 2). In vaccinia virus a complex between the sulfhydryl oxidase E10R and the redox active protein A2.5L probably uses molecular oxygen to oxidize an intermediary protein G4L. G4L in turn generates disulfide bonds in substrate viral proteins (47). Once formed buried disulfide bonds are inaccessible to the reducing pathways in the cytoplasm. Hence, providing folding is sufficiently fast, and there should be minimal futile cycling between oxidation and reduction. By having a catalyst for disulfide bond formation that is localized to the site of viral protein biogenesis via membrane association (46) there is no need to compromise the natural reducing pathways found in the cytoplasm; indeed, maintaining them may be beneficial to attain the maximal yield of native disulfide bonds (Fig. 3).

FIG. 3.

Retention of the reducing pathways in the cytoplasm may aid native disulfide bond formation. Native disulfide bond formation can broadly be split into two distinct steps. The initial step is disulfide bond formation or oxidation of a dithiol to a disulfide (reaction 1). During this step incorrect disulfide bonds may be made, especially if the protein contains multiple cysteine residues. These may be converted into native disulfide bonds either by a direct process of isomerization (reaction 2) or by cycles of reduction/oxidation (reaction 3). The known catalysts of efficient disulfide bond isomerization, PDI (21) or DsbC, both have multiple catalytically active sites—in the case of DsbC formed by homodimerization (33)—and a distinct substrate binding site. None of the verified and putative redox active proteins encoded by nucleocytoplasmic large DNA viruses (17) are known to have this combination, though it is possible that they may form it by homo- and heterodimerization. Hence, retention of the reducing pathways in the cytoplasm may aid native disulfide bond formation by allowing cycles of reduction/oxidation in the case of incorrect disulfide bond formation. Such cycling has recently been shown to facilitate disulfide bond formation in the bacterial periplasm (48) and cycling mediated by glutathione reduction is known to be critical for native disulfide bond formation in mitochondria (4). It is noteworthy that the μ1 protein of the outer capsid of reovirus that forms disulfide bonds in dead or dying cells (37), probably by a noncatalytic process, has only 4 cysteines that is, it probably does not require the action of an isomerization system. In contrast, some of the vaccinia virus proteins that have disulfide bonds formed by the virally encoded cytoplasmic redox system [reviewed in (17)], for example, J5L and G9R have a much larger number of cytoplasmic cysteine residues, with A16L having twenty (5.8% of the total cytoplasmic amino acids for this protein). The possession of such a large number of cysteines in a protein that contains disulfide bonds (38) probably necessitates an efficient isomerization system.

The number of viruses that are known to have their own encoded oxidative folding pathways is small. Most are nucleocytoplasmic large DNA viruses (asfarviridae, iridoviridae, mimiviridae, phycodnaviridae, and poxviridae) that appear to have evolved from a single ancestor (13), with the exception being baculovirus (18), also a large DNA virus. In each, the virally encoded oxidative pathways appear to function in the cytoplasm. This is not surprising since there are host mechanisms that could be and are utilized in other cellular compartments. Each of the virally encoded systems for disulfide formation uses a sulfhydryl oxidase akin to E10R, though with widely differing N- and C-terminal extensions and mechanisms of dimerization also encode one or more proteins that may act directly in the pathway for disulfide bond formation, functionally, but not necessarily structurally, akin to A2.5L and/or G4L [reviewed in (17)]. The crystal structures of several components of the virally encoded systems for disulfide bond formation have recently been solved [see (16, 18) as examples].

Disulfide bonds may also form in capsid proteins of other viruses in the cytoplasm through noncatalytic mechanisms. For example, the μ1 protein of the outer capsid of reovirus forms a disulfide-linked dimer in dead or dying cells (37), a process that is reminiscent of keratinization and the formation of cornified epithelia.

Keratinization

Virally encoded disulfide bond formation is not the only route by which structural disulfide bonds form in the cytoplasm of eukaryotes. Keratins are a diverse family of proteins that form the intermediate filaments in epithelial cells. Epithelial cells form many different types of tissues, two of which are keratinized epithelia, including epithelium of oral mucosa, bile ducts, and cornea; and cornified epithelia that include the outer layer of the skin, the epidermis, hair cortex, hair cuticle, and nail plates [reviewed in (7)]. Both keratinized and cornified epithelia contain large amounts of keratins and keratin-associated proteins (KAPs), with flattened cornified cells (corneocytes) containing up to 80%–90% by mass of keratin filaments [(54) and reviewed in (7, 34)]. Those keratins expressed at high levels in these cells, such as the hard acidic keratins in the follicular epidermis, have up to 5% cysteine residues. Keratinized epithelia and more especially corneocytes are associated with the formation of structure stabilizing intra- and/or interchain disulfide bonds. Similarly, other proteins associated with keratinization, such as loricrin, cornifelin, involucrin, filaggrin, desmoplakin, envoplakin, periplakin, and other KAPs have cysteine residues. These vary from having one or two cysteines (typical for a cytoplasmic protein) up to nearly one-third of the residues being cysteines for some KAPs; for example, human KAP4.1 is 146 amino acids in length of which 49 are cysteine residues. During the processes of keratinization and cornificiation, some of these have been reported to form inter-protein disulfide bonds with keratins and with each other. Many of these are poorly defined, but the agreement in the field is that the formation of disulfide bonds is essential for the final required properties of the epidermis or hair.

There are a number of open questions regarding where disulfide bond formation in keratins and KAPs takes place. A number of proteins associated with cornification, for example elafin, enter the secretory pathway and hence disulfide bonds are formed in these proteins in the ER. There are also reports regarding an extracellular skin associated sulfhydryl oxidase (19, 32) suggesting a significant requirement for disulfide bond formation outside the cell. However, there are also a number of indications that disulfide bond formation also occurs inside the cell. Since most keratins, KAPs and other proteins associated with keratinization are located in the cytoplasm any intracellular disulfide bond formation would have to occur in this compartment. The process of keratin filament formation is best studied connected with the formation of hair, though this should be viewed as a highly specialized process that may not necessarily be representative of what is occurring in other tissues.

In hair, keratin filaments are synthesized and assembled in the normal cytoplasmic reducing environment at the base of the hair follicle. Later in the synthesis, KAPs are deposited between the keratin filaments, and together they form the matrix component of the hair fiber. Next, the reducing environment changes to a more oxidizing one that leads to the formation of disulfide bonds between spatially suitable cysteine residues. This is accompanied by a profound structural reorganization in the filaments [i.e., slippage between the antiparallel strands within the filaments and lateral compaction; (14)]. The trigger and the mechanism for the switch from a reducing to an oxidizing environment are unclear. There are no known catalysts for disulfide bond formation in the cytoplasm of eukaryotes, but it is possible that catalysts from other compartments are retargeted with parallels being drawn to the redistribution of the ER during the acrosomal reaction during spermeogenesis [reviewed in (1)]. However, in the absence of other evidence it is likely that the change in intracellular redox state arises from turning off the reductive pathways. This could be via specific downregulation of the transcription/translation of the enzymes involved in maintaining the reductive pathways or by inactivation of these pathways by cell death or, since the pathways for reducing disulfide bonds are NAD(P)H dependent (see The cytoplasm is a reducing environment), the pathways may be inactivated by compromising NAD(P)H production. Whichever route, or combination of routes, is used to downregulate the activity of the reducing pathways, disulfide bonds will naturally accumulate in proteins through a combination of the action of ROS and other reactive chemical species (see also Engineered oxidative folding pathways).

Protein folding in hyperthermophiles

While disulfide bond formation in the ER and IMS is based on the action of sulfhydryl oxidases that can utilize molecular oxygen, disulfide bond formation in the periplasm of prokaryotes is based on the action of the enzymes that do not directly link to molecular oxygen. These systems are either the Dsb (disulfide bond forming) pathway, with DsbB acting as the oxidant, or based on the action of vitamin K oxidoreductases (VKOR) as the oxidant [reviewed in (9)]. Both DsbB and VKOR use small membrane soluble redox intermediates, such as ubiquinone or menaquinone, which can ultimately feed electrons back into the respiratory chain. DsbB and VKOR are transmembrane proteins, with the core structure of each formed from a four helix bundle. Neither oxidizes folding proteins directly, but rather they go through an intermediate protein, for example, DsbA in the case of DsbB, in a scenario analogous to the use of PDI and Mia40; though in some cases the two proteins are fused, for example, VKOR from Synechococcus is formed from a fusion of a VKOR domain and a thioredoxin-like domain (29).

While structural disulfide bonds are generally not observed in cytoplasmic proteins, there is evidence for the formation of structural disulfide bonds in cytoplasmic proteins of hyperthermophiles including adenylosuccinate lyase from Pyrobaculum aerophilium (53) and DNA polymerase from Thermococcus gorgonarius (23). The formation of such structure stabilizing disulfide bonds is conceptually appealing given the extreme temperatures these organisms grow at; for example, P. aerophilium is an aerobe with optimal growth temperature in excess of 100°C. While no biochemical or proteomic studies have been undertaken on these archaea to determine the precise extent of disulfide formation, 79 cytoplasmic proteins from thermophiles contain at least one disulfide bond (2) and sequence analysis has led to the suggestion that up to 44% of cytoplasmic proteins from these organisms may contain structural disulfide bonds (31). To our knowledge no in vivo studies have been undertaken on the potential reductive pathways in the cytoplasm of these organisms, though some in vitro studies on thermophilic thioredoxin reductases have been reported [e.g., (25, 30)]. However, a database search indicates that all of these organisms have the relevant components for at least one of the two reductive pathways encoded in their genome. While it is possible that some of these may either not be expressed or nonfunctional, or have incorrectly assigned function, it is likely that the oxidative pathways for disulfide bond formation in the hyperthermophiles exist in parallel with the reductive pathways without futile cycling. This would be analogous to viral disulfide bond formation (see Viral protein biogenesis) and engineered oxidative folding pathways (see next section). Indeed the existence of parallel oxidative and reductive pathways may be beneficial (see Viral protein biogenesis and Fig. 3). However, such parallel pathways imply that the oxidative pathway must involve an active catalyst and cannot be a passive process similar to that which may occur during keratinization.

The first component identified in the oxidative pathway in the cytoplasm of hyperthermophiles was Protein Disulfide bond Oxidoreductase [PDO; reviewed in (41)]. The crystal structures of PDO from Pyrococcus furiosus, Aeropyrum pernix, and Aquifex aeolicus have been solved and the enzyme consists of two domains each of which has a thioredoxin-fold and a CXXC active site (8, 40, 44). As such PDO is structurally related to PDI (Fig. 4) and more distantly to DsbA. Functionally, PDO also appears to be closely related to PDI (and DsbA), being able to catalyze thiol-disulfide exchange reactions (40, 42). While all of the oxidases in other compartments (sulfhydryl oxidases, DsbB, VKOR) contain bound cofactors PDO does not and hence PDO most likely plays the same role in the cytoplasm of thermophiles as PDI plays in the ER of eukaryotes, as an intermediary between the oxidase and the folding protein.

FIG. 4.

Structural similarities between protein disulfide oxidoreductases (PDO) and PDI suggest commonality of function. PDO was the first enzyme identified in the oxidative pathway in the cytoplasm of hyperthermophiles. Structurally, PDO looks like a mini-version of PDI being comprised of two domains each with a thioredoxin fold (44) while PDI is comprised of four domains each with a thioredoxin fold that are arranged to form a twisted “U” (52). The first catalytic domain of PDI that is replicated in PDO is shown boxed. PDO is not structurally related to sulfhydryl oxidases such as Ero1p (15) or African swine fever virus pB119L (16) and there is no evidence to indicate that it is functionally related that is, it cannot catalyze de novo disulfide bond formation using molecular oxygen. It is the intermediary protein.

Very recently, our group has identified the putative oxidase in the hyperthermophiles. As part of a study relating to engineered oxidative folding pathways (see next section) we examined all of the DsbB and VKOR homologues from Pfam for potential inversion of membrane topology that is, we looked for transmembrane oxidases whose active sites may be located toward the cytoplasm. No naturally inverted DsbB family members were found, but five species were found that contained two VKOR family members, one of which was predicted to be orientated toward the periplasm and the other toward the cytoplasm (22). All five of these organisms belong to the hyperthermophilic archaea and two of these organisms, A. pernix and P. aerophilium, have been shown to have cytoplasmic proteins that contain disulfide bonds and have the highest potential correlation between cytoplasmic disulfide abundance and hyperthermophilicity (2).

The model that then emerges is that nature has adopted a similar system to catalyze disulfide bond formation in each compartment, specifically the use of an oxidase that can introduce disulfide bonds into an intermediary protein folding catalyst which in turn introduces them into folding proteins (compare Figs. 2 and 5). In the case of the hyperthermophile cytoplasm that combination would be “inverted” VKOR and PDO (Fig. 5B). While no studies have been undertaken to directly confirm this model in a hyperthermophile, we have partially reconstituted the pathway in the cytoplasm of E. coli and shown that the naturally inverted VKOR can efficiently transfer disulfide bonds to cytoplasmically located DsbA (as an artificial intermediary protein), which in turn oxidizes folding proteins (22). This system works with both of the natural reducing pathways present in the E. coli cytoplasm intact.

FIG. 5.

Similarities of the oxidative folding pathway in the periplasm and in the cytoplasm of some hyperthermophiles. (A) Disulfide bond formation in the periplasm of prokaryotes is based on one of the transmembrane enzymes DsbB or vitamin-K dependent oxidoreductase (VKOR) that shuttle electrons from a thioredoxin superfamily member for example, DsbA, which acts as an intermediary protein, to a membrane soluble redox intermediate [reviewed in (9)]. The system is reminiscent of the use of an intermediary protein in the ER and IMS of mitochondria (see figure 2). Some VKOR family members have an additional transmembrane region and are fused to a thioredoxin-like protein [e.g. (29)]. (B) Some hyperthermophiles contain two VKOR family members (22), one of which (VKORo) is orientated with its active site located toward the periplasm, the other which (VKORi) is orientated with its active site located toward the cytoplasm. VKORo probably acts with an unidentified thioredoxin superfamily member to form disulfides in the periplasm, while VKORi probably uses PDO as an intermediary protein to form structural disulfide bonds in folding proteins in the cytoplasm. Arrows indicate electron transfer.

Engineered oxidative folding pathways

An estimated one-third of all human proteins contain disulfide bonds and native disulfide bond formation is the rate limiting step in their biogenesis. The large-scale production of proteins with native disulfide bonds is problematic; hence, considerable work has been undertaken into engineering oxidative folding pathways into the cytoplasm of prokaryotes.

The initial work in this area arose from the identification of the components of the reduction pathways by the systematic removal of components [e.g. (43)]. Removal of both reducing pathways, for example a ΔtrxB/Δgor strain (along with a suppressor mutation in alkyl hydroperoxidase that is required for aerobic growth), resulted in systems that were able to make heterologously expressed recombinant disulfide bonded proteins in the cytoplasm (11). This could be further augmented by the addition of a cytoplasmically targeted disulfide isomerase (3), for example, DsbC from the bacterial periplasm. In ΔtrxB/Δgor strains, cytoplasmic thioredoxins, the trxA and trxC gene products in E. coli, are involved in the formation of disulfide bonds in folding proteins such as alkaline phosphatase, in an apparent reversal of their usual role (50). However, as per PDO in hyperthermophiles, thioredoxins are not de novo catalysts of disulfide bond formation but rather they are potential intermediary proteins. Hence, in the absence of the reducing pathways, thioredoxins that have been oxidized by other metabolic processes (Fig. 6) transfer disulfide bonds to folding proteins as there are no other electron donors that thioredoxin can use. In essence, this mirrors the process that may occur in dying cells during cornification (see Keratinization). Hence, structural disulfide bond formation in ΔtrxB/Δgor strains is relatively slow and/or inefficient.

FIG. 6.

Catalysis of structural disulfide bond formation in the cytoplasm by thioredoxin. (A) The cytoplasm of most wild-type prokaryotes and eukaryotes is a reducing environment with thioredoxin family members being found on one of the two branches (see Fig. 1). The redox state of the active site of thioredoxin is oxidized during a variety of metabolic processes, including reduction of ribonucleotide reductase (RNR), the action of ROS and so on. Both reducing pathways can reduce the active site of thioredoxin maintaining a dynamic equilibrium toward the reduced state. (B) When the reducing pathways are disrupted, for example in a ΔtrxB Δgor E. coli strain or during cornification in the epidermis, thioredoxin is predominantly in the oxidized state. It can only revert to the reduced state by transferring a disulfide to another protein. If the disulfide in this protein is accessible to thioredoxin an equilibrium will be established between the reduced and oxidized states of both proteins. Given the low redox potential of thioredoxin [comparison to other superfamily members in (1a)], it is likely the oxidized state of thioredoxin will predominate. In contrast, if the disulfide in the acceptor protein is not accessible to thioredoxin action, for example, it is a buried structural disulfide formed in a folding protein, no equilibrium will be established and thioredoxin will be reduced.

To circumvent this issue our group recently introduced the concept of adding a catalyst of de novo disulfide bond formation to the cytoplasm. The introduction of the sulfhydryl oxidase Erv1p from Saccharomyces (20) or VKORi from Aeropyrum (22) or inversion of DsbB (22), combined with the use of an appropriate intermediate protein and a catalyst of disulfide bond isomerization, results in efficient structural disulfide bond formation even when both pathways for disulfide bond reduction are present. This mirrors the natural pathways found in multiple cellular compartments, including nucleocytoplasmic large DNA viral protein biogenesis (see Viral protein biogenesis) and disulfide bond formation in the cytoplasm of hyperthermophiles (see Protein folding in hyperthermophiles). However, in both systems competition between productive oxidative folding and reduction is probably diminished either by localization of the sulfhydryl oxidase to the site of substrate protein biogenesis (vaccinia virus) or by having only one pathway for reduction present (Aeropyrum). Neither is the case for our recombinant system and for eukaryotic proteins with multiple disulfides this can result in competition between productive oxidative folding and reduction. However, the combination of Erv1p and a ΔtrxB/Δgor strain obviates this problem (35).

Conclusions

For more than four decades structural disulfide bond formation was thought to be limited to a small subset of cellular compartments that did not include the cytoplasm. While this is the case for the majority of proteins that contain structural disulfides, there are multiple pathways for disulfide bond formation in the cytoplasm. These may be noncatalyzed and result from disruption of the reducing pathways, for example, during keratinization, reovirus capsid formation, or ΔtrxB/Δgor bacterial strains; or they may be catalyzed, for example, nucleocytoplasmic large DNA viruses, some hyperthermophiles, or prokaryotes engineered for disulfide bond formation. While such systems may be in a minority, their medical importance combined with the potential biomedical and industrial importance of efficient production of recombinant disulfide bond containing proteins is such that they deserve more widespread recognition and study.

Footnotes

Abbreviations Used

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